Anti-cd276 polypeptides, proteins, and chimeric antigen receptors

ABSTRACT

Polypeptides and proteins that specifically bind to and immunologically recognize CD276 are disclosed. Chimeric antigen receptors (CARs), anti-CD276 binding moieties, nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the polypeptides and proteins are also disclosed. Methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of U.S. Provisional PatentApplication No. 61/805,001, filed Mar. 25, 2013, which is incorporatedby reference herein in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: one 210,944 Byte ASCII (Text) file named“716393_ST25.txt,” dated Mar. 17, 2014.

BACKGROUND OF THE INVENTION

Cancer is a public health concern. Despite advances in treatments suchas chemotherapy, the prognosis for many cancers, including solid tumors,may be poor. It is estimated that about 559,650 Americans will die fromcancer, corresponding to 1,500 deaths per day (Jemal et al., CA CancerJ. Clin., 57:43-66 (2007)). Accordingly, there exists an unmet need foradditional treatments for cancer, particularly solid tumors.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a polypeptide comprising (i) SEQID NOs: 1-6, (ii) SEQ ID NOs: 11-16, or (iii) SEQ ID NOs: 20-25.

Another embodiment of the invention provides a protein comprising afirst polypeptide chain comprising (i) SEQ ID NOs: 1-3, (ii) SEQ ID NOs:11-13, or (iii) SEQ ID NOs: 20-22 and a second polypeptide chaincomprising (i) SEQ ID NOs: 4-6, (ii) SEQ ID NOs: 14-16, or (iii) SEQ IDNOs: 23-25.

Further embodiments of the invention provide related chimeric antigenreceptors (CARs), anti-CD276 binding moieties, nucleic acids,recombinant expression vectors, host cells, populations of cells,conjugates, and pharmaceutical compositions relating to the polypeptidesand proteins of the invention.

Additional embodiments of the invention provide methods of detecting thepresence of cancer in a mammal and methods of treating or preventingcancer in a mammal.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a graph showing percent lysis of target ⁵¹Cr labeled CHO orCHO-276 cells by effector human T cells transduced with a nucleotidesequence encoding a CAR comprising SEQ ID NO: 42 (CD276.1 secondgeneration, version 1) or a CAR comprising SEQ ID NO: 45 (CD276.17second generation, version 1) at various effector to target ratio (E:T)ratios. Open squares represent percent lysis of target CHO cellsco-cultured with cells expressing SEQ ID NO: 42 (CD276.1 secondgeneration, version 1), closed squares represent percent lysis of targetCHO-276 cells co-cultured with cells expressing SEQ ID NO: 42 (CD276.1second generation, version 1), open circles represent percent lysis oftarget CHO cells co-cultured with cells expressing SEQ ID NO: 45(CD276.17 second generation, version 1), and closed circles representpercent lysis of target CHO-276 cells co-cultured with cells expressingSEQ ID NO: 45 (CD276.17 second generation, version 1) at the indicatedeffector to target ratio (E:T) ratios.

FIG. 2 is a graph showing percent lysis of target cells CHO K1 (opensquares), 5838 (closed circles), CHO 276 (closed squares), or K562(diamonds) by effector human cells transduced with a nucleotide sequenceencoding a CAR comprising SEQ ID NO: 42 (CD276.1 second generation,version 1) at the indicated effector to target ratios.

FIG. 3 is a graph showing percent lysis of target cells CHO K1 (opensquares), 5838 (closed circles), CHO 276 (closed squares), K562(diamonds), or TC71 (triangles) by effector human cells transduced witha nucleotide sequence encoding a CAR comprising SEQ ID NO: 39 (CD276.6second generation, version 1) at the indicated effector to targetratios.

FIG. 4 is a graph showing percent lysis of target cells CHO K1 (opensquares), 5838 (closed circles), CHO 276 (closed squares), K562(diamonds), or TC71 (triangles) by effector human cells transduced witha nucleotide sequence encoding a CAR comprising SEQ ID NO: 45 (CD276.17second generation, version 1) at the indicated effector to targetratios.

FIG. 5 is a graph showing the number of spots measured by interferon(IFN)-gamma ELISPOT assay per million effector T cells cultured alone (Tcells only) or co-cultured with CHO-K1, TC71, CHO-276, K562, or 5838cells. The effector T cells were transduced with a nucleotide sequenceencoding a CAR comprising SEQ ID NO: 39 (CD276.6 second generation,version 1) (grey bars) or SEQ ID NO: 45 (CD276.17 second generation,version 1) (black bars).

FIGS. 6 and 7 are graphs showing the optical density (OD) reading at 450nm as measured in an ELISA binding assay for scFv-Fc fusion proteinscomprising heavy and light chain amino acid sequences as follows: (1)Clone CD276.1 (m851) (comprising a heavy chain comprising SEQ ID NO: 17and a light chain comprising SEQ ID NO: 18) (diamonds), (2) CloneCD276.6 (m856) (comprising a heavy chain comprising SEQ ID NO: 7 and alight chain comprising SEQ ID NO: 8) (squares), or (3) Clone CD276.17(m8517) (comprising a heavy chain comprising SEQ ID NO: 26 and a lightchain comprising SEQ ID NO: 27) (triangles), incubated with human (FIG.6) or mouse (FIG. 7) CD276 at the dilutions indicated.

FIG. 8 is a graph showing the binding affinity (response, (RU)) of CloneCD276.6 (m856) (comprising a heavy chain comprising SEQ ID NO: 7 and alight chain comprising SEQ ID NO: 8) to human CD276 over time (s) asmeasured by surface plasmon resonance at KD=1.3×10⁻⁹ M. The dotted linecorresponds to the raw data and the solid line corresponds to the linegenerated by the software when the fitting was performed to calculatethe KD.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the invention provides polypeptides and proteinscomprising an antigen binding domain of an anti-CD276 antibody. Thepolypeptides and proteins advantageously specifically recognize and bindto CD276 (also known as B7-H3). CD276 is expressed or overexpressed on avariety of human tumors, including pediatric solid tumors and adultcarcinomas. Examples of cancers that express or overexpress CD276include, but are not limited to, neuroblastoma, Ewing's sarcoma,rhabdomyosarcoma, and prostate, ovarian, colorectal, and lung cancers.Without being bound to a particular theory or mechanism, it is believedthat by specifically recognizing and binding to CD276, the inventivepolypeptides and proteins may, advantageously, target CD276-expressingcancer cells. In an embodiment of the invention, the inventivepolypeptides and proteins may elicit an antigen-specific responseagainst CD276. Accordingly, without being bound to a particular theoryor mechanism, it is believed that by eliciting an antigen-specificresponse against CD276, the inventive proteins and polypeptides mayprovide for one or more of the following: targeting and destroyingCD276-expressing cancer cells, reducing or eliminating cancer cells,facilitating infiltration of immune cells and/or effector molecules totumor site(s), and enhancing/extending anti-cancer responses.

The term “polypeptide” as used herein includes oligopeptides and refersto a single chain of amino acids connected by one or more peptide bonds.The polypeptide may comprise one or more variable regions (e.g., twovariable regions) of an antigen binding domain of an anti-CD276antibody, each variable region comprising a complementarity determiningregion (CDR) 1, a CDR2, and a CDR3. Preferably, a first variable regioncomprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, 11,or 20 (CDR1 of first variable region), a CDR2 comprising the amino acidsequence of SEQ ID NO: 2, 12, or 21 (CDR2 of first variable region), anda CDR3 comprising the amino acid sequence of SEQ ID NO: 3, 13, or 22(CDR3 of first variable region), and the second variable regioncomprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, 14,or 23 (CDR1 of second variable region), a CDR2 comprising the amino acidsequence of SEQ ID NO: 5, 15, or 24 (CDR2 of second variable region),and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6, 16, or 25(CDR3 of second variable region). In this regard, the inventivepolypeptide can comprise SEQ ID NOs: 1-3, 4-6, 11-13, 14-16, 20-22,23-25, 1-6, 11-16, or 20-25. Accordingly, an embodiment of the inventionprovides a polypeptide comprising (i) SEQ ID NOs: 1-6, (ii) SEQ ID NOs:11-16, or (iii) SEQ ID NOs: 20-25. Preferably, the polypeptide comprisesthe amino acid sequences of SEQ ID NOs: 20-25.

In an embodiment, the polypeptides each comprise one or more variableregions (e.g., first and second variable regions) of an antigen bindingdomain of an anti-CD276 antibody, each comprising the CDRs as describedabove. The first variable region may comprise SEQ ID NO: 8, 18, or 27.The second variable region may comprise SEQ ID NO: 7, 17, or 26.Accordingly, in an embodiment of the invention, the polypeptidecomprises SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 26, SEQ ID NO: 27, SEQ ID NOs: 7 and 8, SEQ ID NOs: 17 and 18, orSEQ ID NOs: 26 and 27. Preferably, the polypeptide comprises SEQ ID NOs:26 and 27.

In an embodiment of the invention, the variable regions of thepolypeptide may be joined by a linker. The linker may comprise anysuitable amino acid sequence. In an embodiment of the invention, thelinker may comprise SEQ ID NO: 9 or 115.

In an embodiment of the invention, the polypeptide comprises twovariable regions, each comprising the CDRs as described above, with alinker positioned between the two variable regions. In this regard, thepolypeptide may comprise SEQ ID NOs: 10, 19, or 28.

In an embodiment, the polypeptide comprises a leader sequence. Theleader sequence may be positioned at the amino terminus of the lightchain variable region. The leader sequence may comprise any suitableleader sequence. In an embodiment, the leader sequence is a humangranulocyte-macrophage colony-stimulating factor (GM-CSF) receptorsequence. The leader sequence may comprise, for example, SEQ ID NO: 60,61, or 62. In an embodiment of the invention, while the leader sequencemay facilitate expression of the polypeptide on the surface of the cell,the presence of the leader sequence in an expressed polypeptide is notnecessary in order for the polypeptide to function. In an embodiment ofthe invention, upon expression of the polypeptide on the cell surface,the leader sequence may be cleaved off of the polypeptide. Accordingly,in an embodiment of the invention, the polypeptide lacks a leadersequence.

The invention further provides a protein comprising at least one of thepolypeptides described herein. By “protein” is meant a moleculecomprising one or more polypeptide chains.

The protein of the invention can comprise a first polypeptide chaincomprising the amino acid sequences of (i) SEQ ID NOs: 1-3, (ii) SEQ IDNOs: 11-13, or (iii) SEQ ID NOs: 20-22 and a second polypeptide chaincomprising (i) SEQ ID NOs: 4-6, (ii) SEQ ID NOs: 14-16, or (iii) SEQ IDNOs: 23-25. The protein of the invention can, for example, comprise afirst polypeptide chain comprising the amino acid sequence of SEQ ID NO:7, 17, or 26 and a second polypeptide chain comprising the amino acidsequence of SEQ ID NO: 8, 18, or 27. In this regard, the protein maycomprise a first polypeptide chain comprising SEQ ID NO: 7, 17, or 26and a second polypeptide chain comprising SEQ ID NO: 8, 18, or 27.

The protein may further comprise a leader sequence and/or a linker asdescribed herein with respect to other aspects of the invention. In anembodiment, the protein lacks a leader sequence.

The protein of the invention can be, for example, a fusion protein. If,for example, the protein comprises a single polypeptide chain comprising(i) SEQ ID NO: 7, 17, or 26 and (ii) SEQ ID NO: 8, 18, or 27, or if thefirst and/or second polypeptide chain(s) of the protein furthercomprise(s) other amino acid sequences, e.g., an amino acid sequenceencoding an immunoglobulin or a portion thereof, then the inventiveprotein can be a fusion protein. In this regard, the invention alsoprovides a fusion protein comprising at least one of the inventivepolypeptides described herein along with at least one other polypeptide.The other polypeptide can exist as a separate polypeptide of the fusionprotein, or can exist as a polypeptide, which is expressed in frame (intandem) with one of the inventive polypeptides described herein. Theother polypeptide can encode any peptidic or proteinaceous molecule, ora portion thereof, including, but not limited to an immunoglobulin, CD3,CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d,etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. See, for instance, Choi et al., Mol.Biotechnol. 31: 193-202 (2005).

It is contemplated that the polypeptides and proteins of the inventionmay be useful as anti-CD276 binding moieties. In this regard, anembodiment of the invention provides an anti-CD276 binding moietycomprising any of the polypeptides or proteins described herein. In anembodiment of the invention, the anti-CD276 binding moiety comprises anantigen binding portion of any of the polypeptides or proteins describedherein. The antigen binding portion can be any portion that has at leastone antigen binding site. In an embodiment, the anti-CD276 bindingmoiety is a Fab fragment (Fab), F(ab′)₂ fragment, diabody, triabody,tetrabody, single-chain variable region fragment (scFv), ordisulfide-stabilized variable region fragment (dsFv).

In an embodiment, the anti-CD276 binding moiety can be an antibody. Theantibody may be, for example, a recombinant antibody comprising at leastone of the inventive polypeptides described herein. As used herein,“recombinant antibody” refers to a recombinant (e.g., geneticallyengineered) protein comprising at least one of the polypeptides orproteins of the invention and one or more polypeptide chains of anantibody, or a portion thereof. The polypeptide of an antibody, orportion thereof, can be, for example, a constant region of a heavy orlight chain, or an Fe fragment of an antibody, etc. The polypeptidechain of an antibody, or portion thereof, can exist as a separatepolypeptide of the recombinant antibody. Alternatively, the polypeptidechain of an antibody, or portion thereof, can exist as a polypeptide,which is expressed in frame (in tandem) with the polypeptide or proteinof the invention. The polypeptide of an antibody, or portion thereof,can be a polypeptide of any antibody or any antibody fragment.

The antibody of the invention can be any type of immunoglobulin that isknown in the art. For instance, the anti-CD276 binding moiety can be anantibody of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. Theantibody can be monoclonal or polyclonal. The antibody can be anaturally-occurring antibody, e.g., an antibody isolated and/or purifiedfrom a mammal, e.g., mouse, rabbit, goat, horse, chicken, hamster,human, etc. Alternatively, the antibody can be a genetically-engineeredantibody, e.g., a humanized antibody or a chimeric antibody. Theantibody can be in monomeric or polymeric form. Also, the antibody canhave any level of affinity or avidity for CD276.

Methods of testing antibodies for the ability to bind to CD276 are knownin the art and include any antibody-antigen binding assay, such as, forexample, radioimmunoassay (RIA), ELISA, Western blot,immunoprecipitation, and competitive inhibition assays (see, e.g.,Murphy et al., infra, and U.S. Patent Application Publication No.2002/0197266 A1).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Köhler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and Murphy et al.(eds.), Murphy's Immunobiology, 7^(th) Ed., Garland Science, New York,N.Y. (2008)). Alternatively, other methods, such as EBV-hybridomamethods (Haskard and Archer, J. Immunol. Methods, 74 (2), 361-67 (1984),and Roder et al., Methods Enzymol., 121, 140-67 (1986)), andbacteriophage vector expression systems (see, e.g., Huse et al.,Science, 246, 1275-81 (1989)) are known in the art. Further, methods ofproducing antibodies in non-human animals are described in, e.g., U.S.Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. PatentApplication Publication No. 2002/0197266 A1.

Phage display furthermore can be used to generate an antibody. In thisregard, phage libraries encoding antigen-binding variable (V) domains ofantibodies can be generated using standard molecular biology andrecombinant DNA techniques. See, for instance, Green et al. (eds.),Molecular Cloning, A Laboratory Manual, 4^(th) Edition, Cold SpringHarbor Laboratory Press, New York (2012) and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, NY (2007). Phage encoding a variable region with thedesired specificity are selected for specific binding to the desiredantigen, and a complete or partial antibody is reconstituted comprisingthe selected variable domain. Nucleic acid sequences encoding thereconstituted antibody are introduced into a suitable cell line, such asa myeloma cell used for hybridoma production, such that antibodieshaving the characteristics of monoclonal antibodies are secreted by thecell (see, e.g., Murphy et al., supra, Huse et al., supra, and U.S. Pat.No. 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Murphy et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Murphy et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol.,235, 959-973 (1994).

In a preferred embodiment, the anti-CD276 binding moiety is asingle-chain variable region fragment (scFv). A single-chain variableregion fragment (scFv) antibody fragment, which is a truncated Fabfragment including the variable (V) domain of an antibody heavy chainlinked to a V domain of a light antibody chain via a synthetic peptide,can be generated using routine recombinant DNA technology techniques(see, e.g., Murphy et al., supra). Similarly, disulfide-stabilizedvariable region fragments (dsFv) can be prepared by recombinant DNAtechnology (see, e.g., Reiter et al., Protein Engineering, 7, 697-704(1994)). The anti-CD276 binding moieties of the invention, however, arenot limited to these exemplary types of antibody fragments.

Also, the anti-CD276 binding moiety can be modified to comprise adetectable label, such as, for instance, a radioisotope, a fluorophore(e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme(e.g., alkaline phosphatase, horseradish peroxidase), and elementparticles (e.g., gold particles).

Another embodiment of the invention provides chimeric antigen receptors(CARs) comprising: (a) an antigen binding domain comprising any of thepolypeptides or proteins described herein, (b) a transmembrane domain,and (c) an intracellular T cell signaling domain.

A chimeric antigen receptor (CAR) is an artificially constructed hybridprotein or polypeptide containing the antigen binding domains of anantibody (e.g., single chain variable fragment (scFv)) linked to T-cellsignaling domains. Characteristics of CARs include their ability toredirect T-cell specificity and reactivity toward a selected target in anon-MHC-restricted manner, exploiting the antigen-binding properties ofmonoclonal antibodies. The non-MHC-restricted antigen recognition givescells expressing CARs the ability to recognize antigen independent ofantigen processing, thus bypassing a major mechanism of tumor escape.Moreover, when expressed in T-cells, CARs advantageously do not dimerizewith endogenous T cell receptor (TCR) alpha and beta chains.

The phrases “have antigen specificity” and “elicit antigen-specificresponse” as used herein means that the CAR can specifically bind to andimmunologically recognize an antigen, such that binding of the CAR tothe antigen elicits an immune response.

The CARs of the invention have antigen specificity for CD276 (also knownas B7-H3). Without being bound to a particular theory or mechanism, itis believed that by eliciting an antigen-specific response againstCD276, the inventive CARs provide for one or more of the following:targeting and destroying CD276-expressing cancer cells, reducing oreliminating cancer cells, facilitating infiltration of immune cells totumor site(s), and enhancing/extending anti-cancer responses.

An embodiment of the invention provides a CAR comprising an antigenbinding domain of an anti-CD276 antibody. The antigen binding domain ofthe anti-CD276 antibody specifically binds to CD276. The antigen bindingdomain of the CARs may comprise any of the polypeptides or proteinsdescribed herein. In an embodiment of the invention, the CAR comprisesan anti-CD276 single chain variable fragment (scFv). In this regard, apreferred embodiment of the invention provides CARs comprising anantigen-binding domain comprising a single chain variable fragment(scFv) that comprises any of the polypeptides or proteins describedherein.

In a preferred embodiment of the invention, the CAR comprises a heavychain and a light chain each of which comprises a variable regioncomprising a complementarity determining region (CDR) 1, a CDR2, and aCDR3. Preferably, the heavy chain comprises a CDR1 comprising the aminoacid sequence of SEQ ID NO: 1, 11, or 20 (CDR1 of heavy chain), a CDR2comprising the amino acid sequence of SEQ ID NO: 2, 12, or 21 (CDR2 ofheavy chain), and a CDR3 comprising the amino acid sequence of SEQ IDNO: 3, 13, or 22 (CDR3 of heavy chain), and the light chain comprises aCDR1 comprising the amino acid sequence of SEQ ID NO: 4, 14, or 23 (CDR1of light chain), a CDR2 comprising the amino acid sequence of SEQ ID NO:5, 15, or 24 (CDR2 of light chain), and a CDR3 comprising the amino acidsequence of SEQ ID NO: 6, 16, or 25 (CDR3 of light chain). In thisregard, the inventive CAR can comprise SEQ ID NOs: 1-3, 4-6, 11-13,14-16, 20-22, 23-25, 1-6, 11-16, or 20-25. Preferably the CAR comprisesthe amino acid sequences of SEQ ID NOs: 20-25.

The antigen binding domains of the CARs each comprise a light chain anda heavy chain. The light chain may comprise SEQ ID NO: 8, 18, or 27. Theheavy chain may comprise SEQ ID NO: 7, 17, or 26. Accordingly, in anembodiment of the invention, the antigen binding domain comprises SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 26, SEQ IDNO: 27, SEQ ID NOs: 7 and 8, SEQ ID NOs: 17 and 18, or SEQ ID NOs: 26and 27. Preferably, the CAR comprises SEQ ID NOs: 26 and 27.

In an embodiment of the invention, the antigen binding domain of the CARcomprises a light chain and a heavy chain, each comprising the CDRs asdescribed above, with a linker positioned between the light chain andthe heavy chain. The linker may be as described herein with respect toother aspects of the invention. In this regard, the CAR may comprise SEQID NOs: 10, 19, or 28.

In an embodiment, the antigen binding domain of the CAR comprises aleader sequence. The leader sequence may be as described herein withrespect to other aspects of the invention. In an embodiment of theinvention, the CAR lacks a leader sequence.

In an embodiment, the CAR comprises an immunoglobulin constant domain.Preferably, the immunoglobulin domain is a human immunoglobulinsequence. In an embodiment, the immunoglobulin constant domain comprisesan immunoglobulin CH2 and CH3 immunoglobulin G (IgG1) domain sequence(CH2CH3). In this regard, the CAR may comprise an immunoglobulinconstant domain comprising SEQ ID NO: 71. In an embodiment of theinvention, the CAR may comprise an amino acid sequence encoding anantigen binding domain and an immunoglobulin constant domain comprisingany one of SEQ ID NOs: 80, 82, and 84. Without being bound to aparticular theory, it is believed that the CH2CH3 domain extends thebinding motif of the scFv away from the membrane of the CAR-expressingcells and may more accurately mimic the size and domain structure of anative TCR. In some embodiments, the CAR may lack an immunoglobulinconstant domain.

In an embodiment of the invention, the CAR comprises a transmembranedomain. In an embodiment of the invention, the transmembrane domaincomprises i) CD8 and/or ii) CD28. In a preferred embodiment, the CD8 andCD28 are human. The CD8 or CD28 may comprise less than the whole CD8 orCD28, respectively. In this regard, the CAR comprises a transmembranedomain comprising any one or more of a CD8 amino acid sequencecomprising SEQ ID NO: 29, a CD28 amino acid sequence comprising SEQ IDNO: 30, and a CD8 amino acid sequence comprising SEQ ID NO: 31.

In an embodiment of the invention, the CAR comprises an intracellular Tcell signaling domain comprising one or more of i) CD28, ii) CD137, andiii) CD3 zeta (ζ). In a preferred embodiment, the one or more of CD28,CD137, and CD3 zeta are human. CD28 is a T cell marker important in Tcell co-stimulation. CD137, also known as 4-1BB, transmits a potentcostimulatory signal to T cells, promoting differentiation and enhancinglong-term survival of T lymphocytes. CD3ζ associates with TCRs toproduce a signal and contains immunoreceptor tyrosine-based activationmotifs (ITAMs). One or more of CD28, CD137, and CD3 zeta may compriseless than the whole CD28, CD137, or CD3 zeta, respectively. In anembodiment of the invention, intracellular T cell signaling domaincomprises a CD28 amino acid sequence comprising SEQ ID NO: 32 and/or SEQID NO: 35. In another embodiment of the invention, the intracellular Tcell signaling domain comprises a CD137 amino acid sequence comprisingSEQ ID NO: 33 and/or SEQ ID NO: 37. In another embodiment of theinvention, the intracellular T cell signaling domain comprises a CD3zeta amino acid sequence comprising any one or more of SEQ ID NOs: 34,36, and 38.

In an embodiment of the invention, the CAR comprises a transmembranedomain comprising CD28 and an intracellular T cell signaling domaincomprising CD28 and CD3 zeta. In this regard, the CAR may comprise eachof SEQ ID NOs: 30, 35, and 36. In an embodiment, a transmembrane domaincomprising CD28 and an intracellular T cell signaling domain comprisingCD28 and CD3 zeta comprises SEQ ID NO: 73. Preferably, the CAR comprises(a) each of SEQ ID NOs: 1-6, 71, 30, 35, and 36; (b) each of SEQ ID NOs:7, 8, 71, 30, 35, and 36; (c) each of SEQ ID NOs: 10, 71, 30, 35, and36; (d) each of SEQ ID NOs: 11-16, 71, 30, 35, and 36; (e) each of SEQID NOs: 17, 18, 71, 30, 35, and 36; (f) each of SEQ ID NOs: 19, 71, 30,35, and 36; (g) each of SEQ ID NOs: 20-25, 71, 30, 35, and 36; (h) eachof SEQ ID NOs: 26, 27, 71, 30, 35, and 36; or (i) each of SEQ ID NOs:28, 71, 30, 35, and 36.

In an embodiment of the invention, the CAR comprises a transmembranedomain comprising CD8 and an intracellular T cell signaling domaincomprising CD28, CD137, and CD3 zeta. In this regard, the CAR maycomprise each of SEQ ID NOs: 29 and 32-34. In an embodiment, atransmembrane domain comprising CD8 and an intracellular T cellsignaling domain comprising CD28, CD137, and CD3 zeta comprises SEQ IDNO: 75. Preferably, the CAR comprises (a) each of SEQ ID NOs: 1-6, 71,29, and 32-34; (b) each of SEQ ID NOs: 7, 8, 71, 29, and 32-34; (c) eachof SEQ ID NOs: 10, 71, 29, and 32-34; (d) each of SEQ ID NOs: 11-16, 71,29, and 32-34; (e) each of SEQ ID NOs: 17, 18, 71, 29, and 32-34; (f)each of SEQ ID NOs: 19, 71, 29, and 32-34; (g) each of SEQ ID NOs:20-25, 71, 29, and 32-34; (h) each of SEQ ID NOs: 26, 27, 71, 29, and32-34; or (i) each of SEQ ID NOs: 28, 71, 29, and 32-34.

In an embodiment of the invention, the CAR comprises a transmembranedomain comprising CD8 and an intracellular T cell signaling domaincomprising CD137 and CD3 zeta. In this regard, the CAR may comprise eachof SEQ ID NOs: 31, 37, and 38. In an embodiment, a transmembrane domaincomprising CD8 and an intracellular T cell signaling domain comprisingCD137 and CD3 zeta comprises SEQ ID NO: 74. Preferably, the CARcomprises each of (a) each of SEQ ID NOs: 1-6, 71, 31, 37, and 38; (b)each of SEQ ID NOs: 7, 8, 71, 31, 37, and 38; (c) each of SEQ ID NOs:10, 71, 31, 37, and 38; (d) each of SEQ ID NOs: 11-16, 71, 31, 37, and38; (e) each of SEQ ID NOs: 17, 18, 71, 31, 37, and 38; (f) each of SEQID NOs: 19, 71, 31, 37, and 38; (g) each of SEQ ID NOs: 20-25, 71, 31,37, and 38; (h) each of SEQ ID NOs: 26, 27, 71, 31, 37, and 38; or (i)each of SEQ ID NOs: 28, 71, 31, 37, and 38.

Additional embodiments of the invention provide CARs comprising one ormore of any of the amino acid sequences set forth in Tables 1A and 1B.

TABLE 1A Antigen Binding SEQ ID NO: Domain Further Components SEQ ID NO:39 CD276.6 scFv CH2CH3 (CD276.6 CAR- (SEQ ID NO: 10) CD28 transmembranedomain second generation, CD28 and CD3ζ intracellular version 1) T cellsignaling domains SEQ ID NO: 40 CD276.6 scFv CH2CH3 (CD276.6 CAR- (SEQID NO: 10) CD8 transmembrane domain second generation, CD137 and CD3ζintracellular version 2) T cell signaling domains SEQ ID NO: 41 CD276.6scFv CH2CH3 (CD276.6 CAR- (SEQ ID NO: 10) CD8 transmembrane domain thirdgeneration) CD28, CD137, and CD3ζ intracellular T cell signaling domainsSEQ ID NO: 42 CD276.1 scFv CH2CH3 (CD276.1 CAR- (SEQ ID NO: 19) CD28transmembrane domain second generation, CD28 and CD3ζ intracellularversion 1) T cell signaling domains SEQ ID NO: 43 CD276.1 scFv CH2CH3(CD276.1 CAR- (SEQ ID NO: 19) CD8 transmembrane domain secondgeneration, CD137 and CD3ζ intracellular version 2) T cell signalingdomains SEQ ID NO: 44 CD276.1 scFv CH2CH3 (CD276.1 CAR- (SEQ ID NO: 19)CD8 transmembrane domain third generation) CD28, CD137, and CD3ζintracellular T cell signaling domains SEQ ID NO: 45 CD276.17 scFvCH2CH3 (CD276.17 CAR- (SEQ ID NO: 28) CD28 transmembrane domain secondgeneration, CD28 and CD3ζ intracellular version 1) T cell signalingdomains SEQ ID NO: 46 CD276.17 scFv CH2CH3 (CD276.17 CAR- (SEQ ID NO:28) CD8 transmembrane domain second generation, CD137 and CD3ζintracellular version 2) T cell signaling domains SEQ ID NO: 47 CD276.17scFv CH2CH3 (CD276.17 CAR- (SEQ ID NO: 28) CD8 transmembrane domainthird generation) CD28, CD137, and CD3ζ intracellular T cell signalingdomains

TABLE 1B Antigen Binding SEQ ID NO: Domain Further Components SEQ ID NO:122 CD276.6 scFv CD28 transmembrane domain (CD276.6 CAR- (SEQ ID NO: 10)CD28 and CD3ζ intracellular second generation, T cell signaling domainsversion 1) SEQ ID NO: 123 CD276.6 scFv CD8 transmembrane domain (CD276.6CAR- (SEQ ID NO: 10) CD137 and CD3ζ intracellular second generation, Tcell signaling domains version 2) SEQ ID NO: 124 CD276.6 scFv CD8transmembrane domain (CD276.6 CAR- (SEQ ID NO: 10) CD28, CD137, and CD3ζthird generation) intracellular T cell signaling domains SEQ ID NO: 125CD276.1 scFv CD28 transmembrane domain (CD276.1 CAR- (SEQ ID NO: 19)CD28 and CD3ζ intracellular second generation, T cell signaling domainsversion 1) SEQ ID NO: 126 CD276.1 scFv CD8 transmembrane domain (CD276.1CAR- (SEQ ID NO: 19) CD137 and CD3ζ intracellular second generation, Tcell signaling domains version 2) SEQ ID NO: 127 CD276.1 scFv CD8transmembrane domain (CD276.1 CAR- (SEQ ID NO: 19) CD28, CD137, and CD3ζthird generation) intracellular T cell signaling domains SEQ ID NO: 128CD276.17 scFv CD28 transmembrane domain (CD276.17 CAR- (SEQ ID NO: 28)CD28 and CD3ζ intracellular second generation, T cell signaling domainsversion 1) SEQ ID NO: 129 CD276.17 scFv CD8 transmembrane domain(CD276.17 CAR- (SEQ ID NO: 28) CD137 and CD3ζ intracellular secondgeneration, T cell signaling domains version 2) SEQ ID NO: 130 CD276.17scFv CD8 transmembrane domain (CD276.17 CAR- (SEQ ID NO: 28) CD28,CD137, and CD3ζ third generation) intracellular T cell signaling domains

Included in the scope of the invention are functional portions of theinventive polypeptides, proteins, and CARs described herein. The term“functional portion” when used in reference to a polypeptide, protein,or CAR refers to any part or fragment of the polypeptide, protein, orCAR of the invention, which part or fragment retains the biologicalactivity of the polypeptide, protein, or CAR of which it is a part (theparent polypeptide, protein, or CAR). Functional portions encompass, forexample, those parts of a polypeptide, protein, or CAR that retain theability to recognize target cells, or detect, treat, or prevent adisease, to a similar extent, the same extent, or to a higher extent, asthe parent polypeptide, protein, or CAR. In reference to the parentpolypeptide, protein, or CAR, the functional portion can comprise, forinstance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of theparent polypeptide, protein, or CAR.

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parentpolypeptide, protein, or CAR. Desirably, the additional amino acids donot interfere with the biological function of the functional portion,e.g., recognize target cells, detect cancer, treat or prevent cancer,etc. More desirably, the additional amino acids enhance the biologicalactivity, as compared to the biological activity of the parentpolypeptide, protein, or CAR.

Included in the scope of the invention are functional variants of theinventive polypeptides, proteins, or CARs described herein. The term“functional variant” as used herein refers to a polypeptide, protein, orCAR having substantial or significant sequence identity or similarity toa parent polypeptide, protein, or CAR, which functional variant retainsthe biological activity of the polypeptide, protein, or CAR of which itis a variant. Functional variants encompass, for example, those variantsof the polypeptide, protein, or CAR described herein (the parentpolypeptide, protein, or CAR) that retain the ability to recognizetarget cells to a similar extent, the same extent, or to a higherextent, as the parent polypeptide, protein, or CAR. In reference to theparent polypeptide, protein, or CAR, the functional variant can, forinstance, be at least about 30%, about 50%, about 75%, about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99% or more identical in aminoacid sequence to the parent polypeptide, protein, or CAR.

A functional variant can, for example, comprise the amino acid sequenceof the parent polypeptide, protein, or CAR with at least oneconservative amino acid substitution. Alternatively or additionally, thefunctional variants can comprise the amino acid sequence of the parentpolypeptide, protein, or CAR with at least one non-conservative aminoacid substitution. In this case, it is preferable for thenon-conservative amino acid substitution to not interfere with orinhibit the biological activity of the functional variant. Thenon-conservative amino acid substitution may enhance the biologicalactivity of the functional variant, such that the biological activity ofthe functional variant is increased as compared to the parentpolypeptide, protein, or CAR.

Amino acid substitutions of the inventive polypeptides, proteins, orCARs are preferably conservative amino acid substitutions. Conservativeamino acid substitutions are known in the art, and include amino acidsubstitutions in which one amino acid having certain physical and/orchemical properties is exchanged for another amino acid that has thesame or similar chemical or physical properties. For instance, theconservative amino acid substitution can be an acidic/negatively chargedpolar amino acid substituted for another acidic/negatively charged polaramino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chainsubstituted for another amino acid with a nonpolar side chain (e.g.,Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), abasic/positively charged polar amino acid substituted for anotherbasic/positively charged polar amino acid (e.g. Lys, His, Arg, etc.), anuncharged amino acid with a polar side chain substituted for anotheruncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr,Tyr, etc.), an amino acid with a beta-branched side-chain substitutedfor another amino acid with a beta-branched side-chain (e.g., Ile, Thr,and Val), an amino acid with an aromatic side-chain substituted foranother amino acid with an aromatic side chain (e.g., His, Phe, Trp, andTyr), etc.

The polypeptide, protein, or CAR can consist essentially of thespecified amino acid sequence or sequences described herein, such thatother components, e.g., other amino acids, do not materially change thebiological activity of the polypeptide, protein, CAR, functionalportion, or functional variant.

The polypeptides, proteins, or CARs of embodiments of the invention(including functional portions and functional variants) can be of anylength, i.e., can comprise any number of amino acids, provided that thepolypeptides, proteins, or CARs (or functional portions or functionalvariants thereof) retain their biological activity, e.g., the ability tospecifically bind to antigen, detect diseased cells in a mammal, ortreat or prevent disease in a mammal, etc. For example, the polypeptide,protein, or CAR can be about 50 to about 5000 amino acids long, such as50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900,1000 or more amino acids in length.

The polypeptides, proteins, or CARs of embodiments of the invention(including functional portions and functional variants of the invention)can comprise synthetic amino acids in place of one or morenaturally-occurring amino acids. Such synthetic amino acids are known inthe art, and include, for example, aminocyclohexane carboxylic acid,norleucine, α-amino n-decanoic acid, homoserine,S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline,4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine,4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine,phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine,indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, aminomalonic acid, aminomalonic acid monoamide,N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine,ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptane carboxylic acid,α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid,α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

The polypeptides, proteins, or CARs of embodiments of the invention(including functional portions and functional variants) can beglycosylated, amidated, carboxylated, phosphorylated, esterified,N-acylated, cyclized via, e.g., a disulfide bridge, or converted into anacid addition salt and/or optionally dimerized or polymerized.

The polypeptides, proteins, or CARs of embodiments of the invention(including functional portions and functional variants thereof) can beobtained by methods known in the art. The polypeptides, proteins, orCARs may be made by any suitable method of making polypeptides orproteins. Suitable methods of de novo synthesizing polypeptides andproteins are described in references, such as Chan et al., Fmoc SolidPhase Peptide Synthesis, Oxford University Press, Oxford, UnitedKingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., MarcelDekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., OxfordUniversity Press, Oxford, United Kingdom, 2001; and U.S. Pat. No.5,449,752. Also, polypeptides and proteins can be recombinantly producedusing the nucleic acids described herein using standard recombinantmethods. See, e.g., Green et al., supra, and Ausubel et al., supra.Further, some of the polypeptides, proteins, or CARs of the invention(including functional portions and functional variants thereof) can beisolated and/or purified from a source, such as a plant, a bacterium, aninsect, a mammal, e.g., a rat, a human, etc. Methods of isolation andpurification are well-known in the art. Alternatively, the polypeptides,proteins, or CARs described herein (including functional portions andfunctional variants thereof) can be commercially synthesized bycompanies, such as Synpep (Dublin, Calif.), Peptide Technologies Corp.(Gaithersburg, Md.), and Multiple Peptide Systems (San Diego, Calif.).In this respect, the inventive polypeptides, proteins, or CARs can besynthetic, recombinant, isolated, and/or purified.

Included in the scope of the invention are conjugates, e.g.,bioconjugates, comprising any of the inventive polypeptides, proteins,CARs, anti-CD276 binding moieties, conjugates, or functional portions orfunctional variants thereof. Conjugates, as well as methods ofsynthesizing conjugates in general, are known in the art (See, forinstance, Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005) and Kirinet al., Inorg Chem. 44 (15): 5405-5415 (2005)). In this regard, anembodiment of the invention provides a conjugate comprising (a) any ofthe polypeptides, proteins, CARs, anti-CD276 binding moieties describedherein conjugated to (b) an effector molecule. The effector molecule maybe any therapeutic molecule or a molecule that facilitates the detectionof the conjugate. The effector molecule is not limited and may be anysuitable effector molecule. For example, the effector molecule may beany one or more of a drug, toxin, label (e.g., any of the detectablelabels described herein), small molecule, or another antibody. Forexample, the toxin may be Pseudomonas exotoxin A (“PE”) or variantsthereof such as, e.g., any of PE4E, PE40, PE38, PE25, PE38QQR, PE38KDEL,PE-LR, and PE35, as described in, e.g., U.S. Pat. Nos. 4,892,827;5,512,658; 5,602,095; 5,608,039; 5,821,238; 5,854,044; U.S. PatentApplication Publication No. US 2010/0215656; and WO 2012/041234, each ofwhich is incorporated herein by reference.

Further provided by an embodiment of the invention is a nucleic acidcomprising a nucleotide sequence encoding any of the polypeptides,proteins, CARs, anti-CD276 binding moieties, conjugates, or functionalportions or functional variants thereof. The nucleic acids of theinvention may comprise a nucleotide sequence encoding any of the leadersequences, linkers, antigen binding domains, immunoglobulin domains,transmembrane domains, and/or intracellular T cell signaling domainsdescribed herein. For example, the nucleic acids may comprise anucleotide sequence encoding a leader, the nucleotide sequencecomprising SEQ ID NO: 63 or 64. Alternatively or additionally, thenucleic acids may comprise a nucleotide sequence encoding animmunoglobulin constant domain, the nucleotide sequence comprising SEQID NO: 72. Alternatively or additionally, the nucleic acids may comprisea nucleotide sequence encoding a transmembrane domain comprising CD28and an intracellular T cell signaling domain comprising CD28 and CD3zeta, the nucleotide sequence comprising SEQ ID NO: 76. Alternatively oradditionally, the nucleic acids may comprise a nucleotide sequenceencoding a transmembrane domain comprising CD8 and an intracellular Tcell signaling domain comprising CD28, CD137, and CD3 zeta, thenucleotide sequence comprising SEQ ID NO: 78. Alternatively oradditionally, the nucleic acids may comprise a nucleotide sequenceencoding a transmembrane domain comprising CD8 and an intracellular Tcell signaling domain comprising CD137 and CD3 zeta, the nucleotidesequence comprising SEQ ID NO: 77. Alternatively or additionally, thenucleic acids may comprise a nucleotide sequence encoding a leader, anantigen binding domain, and an immunoglobulin domain, the nucleotidessequence comprising SEQ ID NO: 79, 81, or 83.

An embodiment of the invention provides a nucleic acid comprising anucleotide sequence encoding any of the polypeptides, proteins, orantigen binding domains described herein. In this regard, the nucleicacid encoding a polypeptide or protein may comprise a nucleotidesequence comprising SEQ ID NO: 57 (CD276.1 antigen binding domain), SEQID NO: 58 (CD276.6 antigen binding domain), or SEQ ID NO: 59 (CD276.17antigen binding domain). In another embodiment of the invention, thenucleic acid encoding the variable regions of a polypeptide or proteinmay comprise a nucleotide sequence comprising (i) SEQ ID NOs: 116 and117, (ii) SEQ ID NOs: 118 and 119, or (iii) SEQ ID NOs: 120 and 121.Another embodiment of the invention provides a nucleic acid comprising anucleotide sequence encoding any of the CARs described herein. In thisregard, the nucleic acid may comprise one or more of any of thenucleotide sequences set forth in Tables 2A and 2B. Any of the nucleicacids described herein may further comprise, on the 5′ end, a nucleotidesequence encoding a leader sequence comprising, for example, SEQ ID NO:140.

TABLE 2A Antigen Binding SEQ ID NO: Domain Further Components SEQ ID NO:48 CD276.6 scFv CH2CH3 (CD276.6 CAR- (SEQ ID NO: 58) CD28 transmembranedomain second generation, CD28 and CD3ζ intracellular version 1) T cellsignaling domains SEQ ID NO: 49 CD276.6 scFv CH2CH3 (CD276.6 CAR- (SEQID NO: 58) CD8 transmembrane domain second generation, CD137 and CD3ζintracellular version 2) T cell signaling domains SEQ ID NO: 50 CD276.6scFv CH2CH3 (CD276.6 CAR- (SEQ ID NO: 58) CD8 transmembrane domain thirdgeneration) CD28, CD137, and CD3ζ intracellular T cell signaling domainsSEQ ID NO: 51 CD276.1 scFv CH2CH3 (CD276.1 CAR- (SEQ ID NO: 57) CD28transmembrane domain second generation, CD28 and CD3ζ intracellularversion 1) T cell signaling domains SEQ ID NO: 52 CD276.1 scFv CH2CH3(CD276.1 CAR- (SEQ ID NO: 57) CD8 transmembrane domain secondgeneration, CD137 and CD3ζ intracellular version 2) T cell signalingdomains SEQ ID NO: 53 CD276.1 scFv CH2CH3 (CD276.1 CAR- (SEQ ID NO: 57)CD8 transmembrane domain third generation) CD28, CD137, and CD3ζintracellular T cell signaling domains SEQ ID NO: 54 CD276.17 scFvCH2CH3 (CD276.17 CAR- (SEQ ID NO: 59) CD28 transmembrane domain secondgeneration, CD28 and CD3ζ intracellular version 1) T cell signalingdomains SEQ ID NO: 55 CD276.17 scFv CH2CH3 (CD276.17 CAR- (SEQ ID NO:59) CD8 transmembrane domain second generation, CD137 and CD3ζintracellular version 2) T cell signaling domains SEQ ID NO: 56 CD276.17scFv CH2CH3 (CD276.17 CAR- (SEQ ID NO: 59) CD8 transmembrane domainthird generation) CD28, CD137, and CD3ζ intracellular T cell signalingdomains

TABLE 2B Antigen Binding SEQ ID NO: Domain Further Components SEQ ID NO:131 CD276.6 scFv CD28 transmembrane domain (CD276.6 CAR- (SEQ ID NO: 58)CD28 and CD3ζ intracellular second generation, T cell signaling domainsversion 1) SEQ ID NO: 132 CD276.6 scFv CD8 transmembrane domain (CD276.6CAR- (SEQ ID NO: 58) CD137 and CD3ζ intracellular second generation, Tcell signaling domains version 2) SEQ ID NO: 133 CD276.6 scFv CD8transmembrane domain (CD276.6 CAR- (SEQ ID NO: 58) CD28, CD137, and CD3ζthird generation) intracellular T cell signaling domains SEQ ID NO: 134CD276.1 scFv CD28 transmembrane domain (CD276.1 CAR- (SEQ ID NO: 57)CD28 and CD3ζ intracellular second generation, T cell signaling domainsversion 1) SEQ ID NO: 135 CD276.1 scFv CD8 transmembrane domain (CD276.1CAR- (SEQ ID NO: 57) CD137 and CD3ζ intracellular second generation, Tcell signaling domains version 2) SEQ ID NO: 136 CD276.1 scFv CD8transmembrane domain (CD276.1 CAR- (SEQ ID NO: 57) CD28, CD137, and CD3ζthird generation) intracellular T cell signaling domains SEQ ID NO: 137CD276.17 scFv CD28 transmembrane domain (CD276.17 CAR- (SEQ ID NO: 59)CD28 and CD3ζ intracellular second generation, T cell signaling domainsversion 1) SEQ ID NO: 138 CD276.17 scFv CD8 transmembrane domain(CD276.17 CAR- (SEQ ID NO: 59) CD137 and CD3ζ intracellular secondgeneration, T cell signaling domains version 2) SEQ ID NO: 139 CD276.17scFv CD8 transmembrane domain (CD276.17 CAR- (SEQ ID NO: 59) CD28,CD137, and CD3ζ third generation) intracellular T cell signaling domains

“Nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. In some embodiments, the nucleic aciddoes not comprise any insertions, deletions, inversions, and/orsubstitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions. In someembodiments, the nucleic acid may encode additional amino acid sequencesthat do not affect the function of the polypeptide, protein, or CAR andwhich may or may not be translated upon expression of the nucleic acidby a host cell (e.g., AAA). In an embodiment of the invention, thenucleic acid is complementary DNA (cDNA).

The nucleic acids of an embodiment of the invention may be recombinant.As used herein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can consist essentially of the specified nucleotidesequence or sequences described herein, such that other components,e.g., other nucleotides, do not materially change the biologicalactivity of the encoded CAR, polypeptide, protein, functional portion,or functional variant.

A recombinant nucleic acid may be one that has a sequence that is notnaturally occurring or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination is often accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, e.g., by genetic engineering techniques, such as thosedescribed in Green et al., supra. The nucleic acids can be constructedbased on chemical synthesis and/or enzymatic ligation reactions usingprocedures known in the art. See, for example, Green et al., supra, andAusubel et al., supra. For example, a nucleic acid can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedupon hybridization (e.g., phosphorothioate derivatives and acridinesubstituted nucleotides). Examples of modified nucleotides that can beused to generate the nucleic acids include, but are not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid can comprise any isolated or purified nucleotidesequence which encodes any of the polypeptides, proteins, CARs,anti-CD276 binding moieties, conjugates, or functional portions orfunctional variants thereof. Alternatively, the nucleotide sequence cancomprise a nucleotide sequence which is degenerate to any of thesequences or a combination of degenerate sequences.

An embodiment of the invention also provides an isolated or purifiednucleic acid comprising a nucleotide sequence which is complementary tothe nucleotide sequence of any of the nucleic acids described herein ora nucleotide sequence which hybridizes under stringent conditions to thenucleotide sequence of any of the nucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditions mayhybridize under high stringency conditions. By “high stringencyconditions” is meant that the nucleotide sequence specificallyhybridizes to a target sequence (the nucleotide sequence of any of thenucleic acids described herein) in an amount that is detectably strongerthan non-specific hybridization. High stringency conditions includeconditions which would distinguish a polynucleotide with an exactcomplementary sequence, or one containing only a few scatteredmismatches from a random sequence that happened to have a few smallregions (e.g., 3-10 bases) that matched the nucleotide sequence. Suchsmall regions of complementarity are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive polypeptides,proteins, CARs, anti-CD276 binding moieties, conjugates, or functionalportions or functional variants thereof. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide.

The invention also provides a nucleic acid comprising a nucleotidesequence that is at least about 70% or more, e.g., about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to any of the nucleic acidsdescribed herein.

In an embodiment, the nucleic acids of the invention can be incorporatedinto a recombinant expression vector. In this regard, an embodiment ofthe invention provides recombinant expression vectors comprising any ofthe nucleic acids of the invention. For purposes herein, the term“recombinant expression vector” means a genetically-modifiedoligonucleotide or polynucleotide construct that permits the expressionof an mRNA, protein, polypeptide, or peptide by a host cell, when theconstruct comprises a nucleotide sequence encoding the mRNA, protein,polypeptide, or peptide, and the vector is contacted with the cell underconditions sufficient to have the mRNA, protein, polypeptide, or peptideexpressed within the cell. The vectors of the invention are notnaturally-occurring as a whole. However, parts of the vectors can benaturally-occurring. The inventive recombinant expression vectors cancomprise any type of nucleotides, including, but not limited to DNA andRNA, which can be single-stranded or double-stranded, synthesized orobtained in part from natural sources, and which can contain natural,non-natural or altered nucleotides. The recombinant expression vectorscan comprise naturally-occurring or non-naturally-occurringinternucleotide linkages, or both types of linkages. Preferably, thenon-naturally occurring or altered nucleotides or internucleotidelinkages do not hinder the transcription or replication of the vector.

In an embodiment, the recombinant expression vector of the invention canbe any suitable recombinant expression vector, and can be used totransform or transfect any suitable host cell. Suitable vectors includethose designed for propagation and expansion or for expression or both,such as plasmids and viruses. The vector can be selected from the groupconsisting of the pUC series (Fermentas Life Sciences, Glen Burnie,Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pETseries (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech,Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.).Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene),λEMBL4, and λNM1149, also can be used. Examples of plant expressionvectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech).Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo(Clontech). The recombinant expression vector may be a viral vector,e.g., a retroviral vector.

A number of transfection techniques are generally known in the art (see,e.g., Graham et al., Virology, 52: 456-467 (1973); Green et al., supra;Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); andChu et al., Gene, 13: 97 (1981). Transfection methods include calciumphosphate co-precipitation (see, e.g., Graham et al., supra), directmicro injection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al.,BioTechniques, 6: 742-751 (1988)), liposome mediated gene transfer (see,e.g., Mannino et al., BioTechniques, 6: 682-690 (1988)), lipid mediatedtransduction (see, e.g., Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid delivery using high velocitymicroprojectiles (see, e.g., Klein et al., Nature, 327: 70-73 (1987)).

In an embodiment, the recombinant expression vectors of the inventioncan be prepared using standard recombinant DNA techniques described in,for example, Green et al., supra, and Ausubel et al., supra. Constructsof expression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColEl, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

The recombinant expression vector may comprise regulatory sequences,such as transcription and translation initiation and termination codons,which are specific to the type of host cell (e.g., bacterium, fungus,plant, or animal) into which the vector is to be introduced, asappropriate, and taking into consideration whether the vector is DNA- orRNA-based. The recombinant expression vector may comprise restrictionsites to facilitate cloning. Examples of sequences including restrictionsites include SEQ ID NOs: 65-70.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected host cells.Marker genes include biocide resistance, e.g., resistance toantibiotics, heavy metals, etc., complementation in an auxotrophic hostto provide prototrophy, and the like. Suitable marker genes for theinventive expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding thepolypeptides, proteins, CARs, anti-CD276 binding moieties, conjugates,or functional portions or functional variants thereof, or to thenucleotide sequence which is complementary to or which hybridizes to thenucleotide sequence encoding the inventive polypeptides, proteins, CARs,anti-CD276 binding moieties, conjugates, or functional portions orfunctional variants thereof. The selection of promoters, e.g., strong,weak, inducible, tissue-specific and developmental-specific, is withinthe ordinary skill of the artisan. Similarly, the combining of anucleotide sequence with a promoter is also within the ordinary skill ofthe artisan. The promoter can be a non-viral promoter or a viralpromoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, anRSV promoter, or a promoter found in the long-terminal repeat of themurine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression.

Further, the recombinant expression vectors can be made to include asuicide gene. As used herein, the term “suicide gene” refers to a genethat causes the cell expressing the suicide gene to die. The suicidegene can be a gene that confers sensitivity to an agent, e.g., a drug,upon the cell in which the gene is expressed, and causes the cell to diewhen the cell is contacted with or exposed to the agent. Suicide genesare known in the art (see, for example, Suicide Gene Therapy: Methodsand Reviews, Springer, Caroline J. (Cancer Research UK Centre for CancerTherapeutics at the Institute of Cancer Research, Sutton, Surrey, UK),Humana Press, 2004) and include, for example, the Herpes Simplex Virus(HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleosidephosphorylase, and nitroreductase.

An embodiment of the invention further provides a host cell comprisingany of the recombinant expression vectors described herein. As usedherein, the term “host cell” refers to any type of cell that can containthe inventive recombinant expression vector. The host cell can be aeukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5α E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell may be a prokaryoticcell, e.g., a DH5α cell. For purposes of producing a recombinantpolypeptide, protein, CAR, anti-CD276 binding moiety, conjugate, orfunctional portion or functional variant thereof, the host cell may be amammalian cell. The host cell may be a human cell. While the host cellcan be of any cell type, can originate from any type of tissue, and canbe of any developmental stage, the host cell may be a peripheral bloodlymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The hostcell may be a B cell or a T cell.

For purposes herein, the T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. The T cell may be a human T cell. The T cell may be a Tcell isolated from a human. The T cell can be any type of T cell and canbe of any developmental stage, including but not limited to, CD4⁺/CD8⁺double positive T cells, CD4⁺ helper T cells, e.g., Th₁ and Th₂ cells,CD8⁺ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memoryT cells, naïve T cells, and the like. The T cell may be a CD8⁺ T cell ora CD4⁺ T cell.

Also provided by an embodiment of the invention is a population of cellscomprising at least one host cell described herein. The population ofcells can be a heterogeneous population comprising the host cellcomprising any of the recombinant expression vectors described, inaddition to at least one other cell, e.g., a host cell (e.g., a T cell),which does not comprise any of the recombinant expression vectors, or acell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, anerythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, amuscle cell, a brain cell, etc. Alternatively, the population of cellscan be a substantially homogeneous population, in which the populationcomprises mainly host cells (e.g., consisting essentially of) comprisingthe recombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

The polypeptides, proteins, CARs (including functional portions andvariants thereof), nucleic acids, recombinant expression vectors, hostcells (including populations thereof), anti-CD276 binding moieties, andconjugates, all of which are collectively referred to as “inventiveanti-CD276 materials” hereinafter, can be isolated and/or purified. Theterm “isolated” as used herein means having been removed from itsnatural environment. The term “purified” or “isolated” does not requireabsolute purity or isolation; rather, it is intended as a relative term.Thus, for example, a purified (or isolated) host cell preparation is onein which the host cell is more pure than cells in their naturalenvironment within the body. Such host cells may be produced, forexample, by standard purification techniques. In some embodiments, apreparation of a host cell is purified such that the host cellrepresents at least about 50%, for example, at least about 70%, of thetotal cell content of the preparation. For example, the purity can be atleast about 50%, can be greater than about 60%, about 70% or about 80%,or can be about 100%.

The inventive anti-CD276 materials can be formulated into a composition,such as a pharmaceutical composition. In this regard, an embodiment ofthe invention provides a pharmaceutical composition comprising any ofthe inventive anti-CD276 materials described herein and apharmaceutically acceptable carrier. The inventive pharmaceuticalcompositions containing any of the inventive anti-CD276 materials cancomprise more than one inventive anti-CD276 material, e.g., a CAR and anucleic acid, or two or more different CARs. Alternatively, thepharmaceutical composition can comprise an inventive CAR material incombination with other pharmaceutically active agents or drugs, such aschemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin,cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine,hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine,vincristine, etc. In a preferred embodiment, the pharmaceuticalcomposition comprises the inventive host cell or populations thereof.

The inventive anti-CD276 materials can be provided in the form of asalt, e.g., a pharmaceutically acceptable salt. Suitablepharmaceutically acceptable acid addition salts include those derivedfrom mineral acids, such as hydrochloric, hydrobromic, phosphoric,metaphosphoric, nitric, and sulphuric acids, and organic acids, such astartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic,gluconic, succinic, and arylsulphonic acids, for example,p-toluenesulphonic acid.

With respect to pharmaceutical compositions, the pharmaceuticallyacceptable carrier can be any of those conventionally used and islimited only by chemico-physical considerations, such as solubility andlack of reactivity with the active agent(s), and by the route ofadministration. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active agent(s) and one which hasno detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive CAR material, as well as by the particular method used toadminister the inventive CAR material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. Preservatives may be used. Suitable preservatives mayinclude, for example, methylparaben, propylparaben, sodium benzoate, andbenzalkonium chloride. A mixture of two or more preservatives optionallymay be used. The preservative or mixtures thereof are typically presentin an amount of about 0.0001% to about 2% by weight of the totalcomposition.

Suitable buffering agents may include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. A mixture of two or more buffering agents optionally may beused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition.

The concentration of inventive anti-CD276 material in the pharmaceuticalformulations can vary, e.g., from less than about 1%, usually at or atleast about 10%, to as much as, for example, about 20% to about 50% ormore by weight, and can be selected primarily by fluid volumes, andviscosities, in accordance with the particular mode of administrationselected.

Methods for preparing administrable (e.g., parenterally administrable)compositions are known or apparent to those skilled in the art and aredescribed in more detail in, for example, Remington: The Science andPractice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1,2005).

The following formulations for oral, aerosol, parenteral (e.g.,subcutaneous, intravenous, intraarterial, intramuscular, intradermal,interperitoneal, and intrathecal), and topical administration are merelyexemplary and are in no way limiting. More than one route can be used toadminister the inventive anti-CD276 materials, and in certain instances,a particular route can provide a more immediate and more effectiveresponse than another route.

Formulations suitable for oral administration can comprise or consist of(a) liquid solutions, such as an effective amount of the inventiveanti-CD276 material dissolved in diluents, such as water, saline, ororange juice; (b) capsules, sachets, tablets, lozenges, and troches,each containing a predetermined amount of the active ingredient, assolids or granules; (c) powders; (d) suspensions in an appropriateliquid; and (e) suitable emulsions. Liquid formulations may includediluents, such as water and alcohols, for example, ethanol, benzylalcohol, and the polyethylene alcohols, either with or without theaddition of a pharmaceutically acceptable surfactant. Capsule forms canbe of the ordinary hard or softshelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers, such as lactose,sucrose, calcium phosphate, and corn starch. Tablet forms can includeone or more of lactose, sucrose, mannitol, corn starch, potato starch,alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, calcium stearate, zinc stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, disintegratingagents, moistening agents, preservatives, flavoring agents, and otherpharmacologically compatible excipients. Lozenge forms can comprise theinventive anti-CD276 material in a flavor, usually sucrose and acacia ortragacanth, as well as pastilles comprising the inventive anti-CD276material in an inert base, such as gelatin and glycerin, or sucrose andacacia, emulsions, gels, and the like containing, in addition to, suchexcipients as are known in the art.

Formulations suitable for parenteral administration include aqueous andnonaqueous isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and nonaqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The inventive anti-CD276 material can be administered in aphysiologically acceptable diluent in a pharmaceutical carrier, such asa sterile liquid or mixture of liquids, including water, saline, aqueousdextrose and related sugar solutions, an alcohol, such as ethanol orhexadecyl alcohol, a glycol, such as propylene glycol or polyethyleneglycol, dimethylsulfoxide, glycerol, ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol) 400,oils, fatty acids, fatty acid esters or glycerides, or acetylated fattyacid glycerides with or without the addition of a pharmaceuticallyacceptable surfactant, such as a soap or a detergent, suspending agent,such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations will typically contain, for example, fromabout 0.5% to about 25% by weight of the inventive anti-CD276 materialin solution. Preservatives and buffers may be used. In order to minimizeor eliminate irritation at the site of injection, such compositions maycontain one or more nonionic surfactants having, for example, ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations will typically range, forexample, from about 5% to about 15% by weight. Suitable surfactantsinclude polyethylene glycol sorbitan fatty acid esters, such as sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol. The parenteral formulations can be presented inunit-dose or multi-dose sealed containers, such as ampoules and vials,and can be stored in a freeze-dried (lyophilized) condition requiringonly the addition of the sterile liquid excipient, for example, water,for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions can be prepared from sterile powders,granules, and tablets of the kind previously described.

Injectable formulations are in accordance with an embodiment of theinvention. The requirements for effective pharmaceutical carriers forinjectable compositions are well-known to those of ordinary skill in theart (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. LippincottCompany, Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250(1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages622-630 (1986)).

Topical formulations, including those that are useful for transdermaldrug release, are well known to those of skill in the art and aresuitable in the context of embodiments of the invention for applicationto skin. The inventive anti-CD276 material, alone or in combination withother suitable components, can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer. Such spray formulations also may be usedto spray mucosa.

An “effective amount” or “an amount effective to treat” refers to a dosethat is adequate to prevent or treat cancer in an individual. Amountseffective for a therapeutic or prophylactic use will depend on, forexample, the stage and severity of the disease or disorder beingtreated, the age, weight, and general state of health of the patient,and the judgment of the prescribing physician. The size of the dose willalso be determined by the active selected, method of administration,timing and frequency of administration, the existence, nature, andextent of any adverse side-effects that might accompany theadministration of a particular active, and the desired physiologicaleffect. It will be appreciated by one of skill in the art that variousdiseases or disorders could require prolonged treatment involvingmultiple administrations, perhaps using the inventive anti-CD276materials in each or various rounds of administration. By way of exampleand not intending to limit the invention, the dose of the inventiveanti-CD276 material can be about 0.001 to about 1000 mg/kg body weightof the subject being treated/day, from about 0.01 to about 10 mg/kg bodyweight/day, about 0.01 mg to about 1 mg/kg body weight/day. When theinventive anti-CD276 material is a host cell, an exemplary dose of hostcells may be a minimum of one million cells (1 mg cells/dose). When theinventive anti-CD276 material is a nucleic acid packaged in a virus, anexemplary dose of virus may be 1 ng/dose.

For purposes of the invention, the amount or dose of the inventiveanti-CD276 material administered should be sufficient to effect atherapeutic or prophylactic response in the subject or animal over areasonable time frame. For example, the dose of the inventive anti-CD276material should be sufficient to bind to antigen, or detect, treat orprevent disease in a period of from about 2 hours or longer, e.g., about12 to about 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive anti-CD276material and the condition of the animal (e.g., human), as well as thebody weight of the animal (e.g., human) to be treated.

For purposes of the invention, an assay, which comprises, for example,comparing the extent to which target cells are lysed and/or IFN-γ issecreted by T cells expressing the inventive anti-CD276 material uponadministration of a given dose of such T cells to a mammal, among a setof mammals of which is each given a different dose of the T cells, couldbe used to determine a starting dose to be administered to a mammal. Theextent to which target cells are lysed and/or IFN-γ is secreted uponadministration of a certain dose can be assayed by methods known in theart.

In addition to the aforedescribed pharmaceutical compositions, theinventive anti-CD276 materials can be formulated as inclusion complexes,such as cyclodextrin inclusion complexes, or liposomes. Liposomes canserve to target the inventive anti-CD276 materials to a particulartissue. Liposomes also can be used to increase the half-life of theinventive anti-CD276 materials. Many methods are available for preparingliposomes, as described in, for example, Szoka et al., Ann. Rev.Biophys. Bioeng., 9, 467 (1980) and U.S. Pat. Nos. 4,235,871, 4,501,728,4,837,028, and 5,019,369.

The delivery systems useful in the context of embodiments of theinvention may include time-released, delayed release, and sustainedrelease delivery systems such that the delivery of the inventivecomposition occurs prior to, and with sufficient time to cause,sensitization of the site to be treated. The inventive composition canbe used in conjunction with other therapeutic agents or therapies. Suchsystems can avoid repeated administrations of the inventive composition,thereby increasing convenience to the subject and the physician, and maybe particularly suitable for certain composition embodiments of theinvention.

Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are lipids including sterols suchas cholesterol, cholesterol esters, and fatty acids or neutral fats suchas mono-di-and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the active composition is contained in a form within amatrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014,4,748,034, and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,832,253 and 3,854,480. In addition,pump-based hardware delivery systems can be used, some of which areadapted for implantation.

One of ordinary skill in the art will readily appreciate that theinventive anti-CD276 materials of the invention can be modified in anynumber of ways, such that the therapeutic or prophylactic efficacy ofthe inventive anti-CD276 materials is increased through themodification. For instance, the inventive anti-CD276 materials can beconjugated either directly or indirectly through a bridge to a targetingmoiety. The practice of conjugating compounds, e.g., inventiveanti-CD276 materials, to targeting moieties is known in the art. See,for instance, Wadwa et al., J. Drug Targeting 3: 111 (1995) and U.S.Pat. No. 5,087,616.

Alternatively, the inventive anti-CD276 materials can be modified into adepot form, such that the manner in which the inventive anti-CD276materials is released into the body to which it is administered iscontrolled with respect to time and location within the body (see, forexample, U.S. Pat. No. 4,450,150). Depot forms of inventive anti-CD276materials can be, for example, an implantable composition comprising theinventive anti-CD276 materials and a porous or non-porous material, suchas a polymer, wherein the inventive anti-CD276 materials areencapsulated by or diffused throughout the material and/or degradationof the non-porous material. The depot is then implanted into the desiredlocation within the body and the inventive anti-CD276 materials arereleased from the implant at a predetermined rate.

When the inventive anti-CD276 materials are administered with one ormore additional therapeutic agents, one or more additional therapeuticagents can be coadministered to the mammal. By “coadministering” ismeant administering one or more additional therapeutic agents and theinventive CAR materials sufficiently close in time such that theinventive anti-CD276 materials can enhance the effect of one or moreadditional therapeutic agents, or vice versa. In this regard, theinventive anti-CD276 materials can be administered first and the one ormore additional therapeutic agents can be administered second, or viceversa. Alternatively, the inventive anti-CD276 materials and the one ormore additional therapeutic agents can be administered simultaneously.An exemplary therapeutic agent that can be co-administered with theanti-CD276 materials is IL-2. It is believed that IL-2 enhances thetherapeutic effect of the inventive anti-CD276 materials. For purposesof the inventive methods, wherein host cells or populations of cells areadministered to the mammal, the cells can be cells that are allogeneicor autologous to the mammal.

It is contemplated that the inventive anti-CD276 materials andpharmaceutical compositions can be used in methods of treating orpreventing a disease in a mammal. Without being bound to a particulartheory or mechanism, the inventive anti-CD276 materials have biologicalactivity, e.g., ability to recognize antigen, e.g., CD276, such that theanti-CD276 material, when expressed by a cell, is able to mediate animmune response against the cell expressing the antigen, e.g., CD276,for which the anti-CD276 material is specific. In this regard, anembodiment of the invention provides a method of treating or preventingcancer in a mammal, comprising administering to the mammal any of thepolypeptides, proteins, CARs, functional portions, functional variants,nucleic acids, recombinant expression vectors, host cells, population ofcells, anti-CD276 binding moieties, conjugates, and/or thepharmaceutical compositions of the invention in an amount effective totreat or prevent cancer in the mammal.

An embodiment of the invention further comprises lymphodepleting themammal prior to administering the inventive anti-CD276 materials.Examples of lymphodepletion include, but may not be limited to,nonmyeloablative lymphodepleting chemotherapy, myeloablativelymphodepleting chemotherapy, total body irradiation, etc.

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

The mammal referred to herein can be any mammal. As used herein, theterm “mammal” refers to any mammal, including, but not limited to,mammals of the order Rodentia, such as mice and hamsters, and mammals ofthe order Logomorpha, such as rabbits. The mammals may be from the orderCarnivora, including Felines (cats) and Canines (dogs). The mammals maybe from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). Themammals may be of the order Primates, Ceboids, or Simoids (monkeys) orof the order Anthropoids (humans and apes). Preferably, the mammal is ahuman.

With respect to the inventive methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer,brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus,anal canal, or anorectum, cancer of the eye, cancer of the intrahepaticbile duct, cancer of the joints, cancer of the neck, gallbladder, orpleura, cancer of the nose, nasal cavity, or middle ear, cancer of theoral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronicmyeloid cancer, colon cancer, Ewing's sarcoma, esophageal cancer,cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, headand neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkinlymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia,liquid tumors, liver cancer, lung cancer (e.g., non-small cell lungcarcinoma), lymphoma, malignant mesothelioma, mastocytoma, melanoma,multiple myeloma, nasopharynx cancer, neuroblastoma, non-Hodgkinlymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acutelymphocytic leukemia (ALL), and Burkitt's lymphoma, ovarian cancer,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, solid tumors, stomach cancer,testicular cancer, thyroid cancer, and ureter cancer. Preferably, thecancer is a pediatric solid tumor, adult carcinoma, neuroblastoma,Ewing's sarcoma, rhabdomyosarcoma, and prostate, ovarian, colorectal, orlung cancer. Preferably, the cancer is characterized by the expressionor overexpression of CD276.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

Another embodiment of the invention provides a use of any of thepolypeptides, proteins, CARs, functional portions, functional variants,nucleic acids, recombinant expression vectors, host cells, population ofcells, anti-CD276 binding moieties, conjugates, or pharmaceuticalcompositions of the invention for the treatment or prevention of cancerin a mammal.

Another embodiment of the invention provides a method of detecting thepresence of cancer in a mammal, comprising: (a) contacting a samplecomprising one or more cells from the mammal with any of thepolypeptides, proteins, CARs, functional portions, functional variants,nucleic acids, recombinant expression vectors, host cells, population ofcells, anti-CD276 binding moieties, or conjugates of the invention,thereby forming a complex, (b) and detecting the complex, whereindetection of the complex is indicative of the presence of cancer in themammal.

The sample may be obtained by any suitable method, e.g., biopsy ornecropsy. A biopsy is the removal of tissue and/or cells from anindividual. Such removal may be to collect tissue and/or cells from theindividual in order to perform experimentation on the removed tissueand/or cells. This experimentation may include experiments to determineif the individual has and/or is suffering from a certain condition ordisease-state. The condition or disease may be, e.g., cancer.

With respect to an embodiment of the inventive method of detecting thepresence of cancer in a mammal, the sample comprising cells of themammal can be a sample comprising whole cells, lysates thereof, or afraction of the whole cell lysates, e.g., a nuclear or cytoplasmicfraction, a whole protein fraction, or a nucleic acid fraction. If thesample comprises whole cells, the cells can be any cells of the mammal,e.g., the cells of any organ or tissue, including blood cells orendothelial cells.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive CARs, polypeptides,proteins, functional portions, functional variants, nucleic acids,recombinant expression vectors, host cells, populations of cells,anti-CD276 binding moieties, or conjugates, described herein, can belabeled with a detectable label such as, for instance, a radioisotope, afluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase),and element particles (e.g., gold particles).

Methods of testing an anti-CD276 material for the ability to recognizetarget cells and for antigen specificity are known in the art. Forinstance, Clay et al., J. Immunol., 163: 507-513 (1999), teaches methodsof measuring the release of cytokines (e.g., interferon-γ,granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosisfactor a (TNF-α) or interleukin 2 (IL-2)). In addition, anti-CD276material function can be evaluated by measurement of cellularcytoxicity, as described in Zhao et al., J. Immunol., 174: 4415-4423(2005).

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLE 1

This example demonstrates the identification, purification, andcharacterization of anti-CD276 binding domains.

Yeast Display Naïve Human Antibody Library, Antibodies, BiotinylationKit, Cells

A large yeast display naïve single chain variable fragment (scFv) humanantibody library was constructed using a collection of human antibodygene repertoires, including the genes used for the construction of aphage display Fab library (Zhu et al., Methods Mol. Biol., 525: 129-142,xv (2009)).

Mouse monoclonal anti-c-Myc antibody was purchased from Roche(Pleasanton, Calif.). PE-conjugated streptavidin and Alexa-488conjugated goat anti-mouse antibody were purchased from Invitrogen(Carlsbad, Calif.). Protein G columns were purchased from GE healthcare(Waukesha, Wis.). Avi-tag specific biotinylation kits were purchasedfrom Avidity (Aurora, Colo.). Yeast plasmid extraction kits werepurchased from Zymo Research (Irvine, Calif.). 293 free style proteinexpression kits were purchased from Invitrogen. An AutoMACS System waspurchased from Miltenyi Biotec (Cologne, Germany).

MACS Sorting Downsize of the Initial Yeast Display Human AntibodyLibrary

Biotinylated human and mouse CD276 extracellular domain was used as thetarget for three rounds of sorting to downsize the initial yeast displaynaive human antibody library. Approximately 1010 cells from the initialnaïve antibody library and 10 μg of biotinylated CD276 ecto-domain wereincubated in 50 ml PBSA (phosphate-buffered saline containing 0.1%bovine serum albumin) at room temperature (RT) for 2 hr with rotation.The mixture of biotinylated CD276 ecto-domain bound to displayedantibody on cells from the library was washed three times with PBSA andincubated with 100 μl of streptavidin conjugated microbeads at RT fromMiltenyi Biotec. The resultant mixture was washed once with PBSA andloaded onto the AutoMACS system for the first round of sorting. Thesorted cells were amplified in SDCAA media (20 g dextrose, 6.7 g DIFCOyeast nitrogen base without (w/o) amino acids, 5 g BACTO casamino acids,5.4 g Na₂HPO₄ and 8.56 g NaH₂PO₄.H₂O in 1 liter water) at 30° C. and 250revolutions per minute (rpm) for 24 hours (hr). The culture was theninduced in SGCAA media (20 g galactose, 20 g raffinose, 1 g dextrose,6.7 g DIFCO yeast nitrogen base w/o amino acids, 5 g BACTO casaminoacids, 5.4 g Na₂HPO₄ and 8.56 g NaH₂PO₄.H₂O in 1 liter water) at 20° C.and 250 rpm for 16-18 hr.

The same amounts of antigen and incubation volume were used for the nexttwo rounds of sorting. The cell numbers used for these two rounds ofsorting were set at 100 times the size of the sorted pool from theprevious round of sorting to keep the diversity of each sorted pool.

Expression and Purification of scFv-Fc Proteins

Plasmids were extracted from the identified yeast clones using yeastplasmid extraction kits (Zymo Research, Irvine, Calif.), following themanufacturer's instructions. Extracted plasmids were transformed into10G chemical competent E. coli (Lucigen, Middleton, Wis.) for furtheramplification. Plasmids extracted from the bacteria were used for DNAsequencing to obtain the nucleic acid sequences encoding the positivebinder antibodies.

Three anti-CD276 antibodies were identified, each comprising heavy andlight chain amino acid sequences as follows: (1) Clone CD276.1 (m851)(comprising a heavy chain comprising SEQ ID NO: 17 and a light chaincomprising SEQ ID NO: 18), (2) Clone CD276.6 (m856) (comprising a heavychain comprising SEQ ID NO: 7 and a light chain comprising SEQ ID NO:8), and (3) Clone CD276.17 (m8517) (comprising a heavy chain comprisingSEQ ID NO: 26 and a light chain comprising SEQ ID NO: 27).

The scFv-encoding inserts of the unique clones were digested with SfiIand ligated into modified pSecTag bearing the same set of SfiI sites andFc-Avi tag for soluble expression. These constructs were transfectedinto 293T cells for expression following the manufacturer's protocol.After 72 hr of growth, the scFv-Fc fusion proteins in the culture mediumwere used for the ELISA binding assay.

ELISA Binding Assay

50 μl of the diluted mouse or human CD276-AP fusion protein in PBS at 2μg/ml was coated in a 96-well plate at 4° C. overnight. Transientlyexpressed scFv-Fc fusion protein in the culture medium was seriallydiluted and added into the target protein coated wells. After washing, a1:3000 diluted horseradish peroxidase (HRP) conjugated goat anti-humanIgG antibody was added for 1 hr at RT. After washing, 3, 3, 5,5′-Tetramethylbenzidine (TMB) substrate was added, and the opticaldensity (O.D.) was read at 450 nm. The results are shown in FIGS. 6 and7. As shown in FIG. 6, scFv-Fc fusion proteins comprising heavy andlight chain amino acid sequences as follows: (1) Clone CD276.1 (m851)(comprising a heavy chain comprising SEQ ID NO: 17 and a light chaincomprising SEQ ID NO: 18), (2) Clone CD276.6 (m856) (comprising a heavychain comprising SEQ ID NO: 7 and a light chain comprising SEQ ID NO:8), and (3) Clone CD276.17 (m8517) (comprising a heavy chain comprisingSEQ ID NO: 26 and a light chain comprising SEQ ID NO: 27) bound to humanCD276. As shown in FIG. 7, all the clones except for m851 (CD276.1)showed cross reactivity to both human and mouse CD276, while m851(CD276.1) is human CD276-specific.

Affinity Determination by Surface Plasmon Resonance

Binding affinities of human anti-CD276 scFv CD276.6 to human CD276Ecto-domain were analyzed by surface plasmon resonance technology usinga Biacore X100 instrument (GE healthcare). The human CD276 solubleextracellular domain was covalently immobilized onto a sensor chip (CM5)using carbodiimide coupling chemistry. A control reference surface wasprepared for nonspecific binding and refractive index changes. Foranalysis of the kinetics of interactions, varying concentrations ofantigens were injected at a flow rate of 30 μl/min using running buffercontaining 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% SurfactantP-20 (pH 7.4). The association and dissociation phase data were fittedsimultaneously to a 1:1 Langumir global model, using the nonlinear dataanalysis program BIAevaluation 3.2. All of the experiments were done at25° C. The affinity of the antibody CD276.6 (m856) (comprising a heavychain comprising SEQ ID NO: 7 and a light chain comprising SEQ ID NO: 8)is shown in FIG. 8.

EXAMPLE 2

This example demonstrates the activity of chimeric antigen receptors(CARs) comprising anti-CD276 binding domains identified in Example 1.

Chimeric antigen receptors (CARs) (second generation-version 1) wereproduced including single chain variable fragments comprising theanti-CD276 binding domains identified in Example 1. In addition to theanti-CD276 binding domains, the CARs also included an immunoglobulindomain comprising an immunoglobulin CH2 and CH3 immunoglobulin G (IgG1)domain sequence (CH2CH3), a CD28 transmembrane domain, a CD28intracellular T cell signaling domain, and a CD3ζ intracellular T cellsignaling domain. The full length sequences of these CARs comprised SEQID NO: 39 (CD276.6 second generation, version 1), SEQ ID NO: 42 (CD276.1second generation, version 1), or SEQ ID NO: 45 (CD276.17 secondgeneration, version 1).

These CARs were tested against tumor cell lines expressing CD276 as wellas a laboratory cell line, CHO, that was transfected to permanentlyexpress either CD276 or a control vector, serving as a positive control(CHO-276) and a negative control (CHO), respectively, in assays ofimmunological function of CAR-transduced T cells. To test CAR activity,human T cells were activated with anti-CD3/CD28 beads in the presence ofinterleukin (IL)-2. These T cells were then transduced with retroviralCAR expression vectors and tested for activity against tumor cell linesin a chromium release assay. The transduced T cells (effectors) werecultured with chromium-labeled target cells CHO or CHO-276 at theeffector to target ratios (number of effector T cells per target cell inthe assay well) shown in FIG. 1. The percentage of target cells thatwere lysed was measured. The results are shown in FIG. 1. The activitiesof cells expressing SEQ ID NO: 42 (CD276.1 second generation, version 1)or SEQ ID NO: 45 (CD276.17 second generation, version 1) are shown inFIG. 1.

As shown in FIG. 1, while both the CAR comprising SEQ ID NO: 42 (CD276.1second generation, version 1) and the CAR comprising SEQ ID NO: 45(CD276.17 second generation, version 1) were active against CHO-276 (theCD276-expressing, positive control cell line), the CAR comprising SEQ IDNO: 45 (CD276.17 second generation, version 1) showed very low activityagainst the control cell line, indicating a high degree of specificlytic activity.

Human cells transduced with nucleotide sequences encoding SEQ ID NO: 39(CD276.6 second generation, version 1), SEQ ID NO: 42 (CD276.1 secondgeneration, version 1), or SEQ ID NO: 45 (CD276.17 second generation,version 1) CARs (effectors) were co-cultured with chromium-labeledtarget CHO K1 cells (negative control), positive control CHO-276 cells,or one of the CD276⁺ tumor cell lines K562 (leukemia cell line), 5838(patient-derived sarcoma cell line), and TC71 (patient-derived sarcomacell line) at the effector to target ratios shown in FIGS. 2-4. Thepercentage of target cells that were lysed was measured. The results areshown in FIGS. 2-4.

As shown in FIGS. 2-4, all CARs were active against CHO-276 (thepositive control) and the 5838 Ewing sarcoma cell line. However, CARsdiffered in their reactivity to the control cell line, with SEQ ID NO:42 (CD276.1 second generation, version 1) and SEQ ID NO: 39 (CD276.6second generation, version 1) showing moderate reactivity to CHO-K1, andSEQ ID NO: 45 (CD276.17 second generation, version 1) showing very lowactivity. Thus, the CD276.17 second generation, version 1 CAR has thehighest degree of specific lytic activity.

The production of interferon (IFN)-gamma was tested in an enzyme-linkedimmunosorbent spot (ELISPOT) assay. In the ELISPOT assay, CAR-transducedT cells were co-incubated with tumor cells for 24 hours and the numberof IFN-gamma producing cells was enumerated by the capture of IFN-gammaon the micro-well filter upon which the assay was performed. Thus, theELISPOT assay both verifies the production of IFN-gamma byCAR-expressing T cells and quantifies the number of T cells in theculture that are able to express IFN-gamma. The results obtained forcells transduced with nucleotide sequences encoding SEQ ID NO: 45(CD276.17 second generation, version 1) or SEQ ID NO: 39 (CD276.6 secondgeneration, version 1) are shown in FIG. 5. As shown in FIG. 5, theCD276.17 second generation, version 1 CAR showed a high degree ofspecific activity in that the greatest number of spots was seen whenCAR-transduced T cells were incubated with the CD276-positive cellslines K562 and 5838 and the positive control cell line, CHO-CD276. Lownumbers of spots were seen with the negative control cell line or when Tcells were incubated alone. Lower levels of activity were seen againstthe TC71 tumor cell line. As in the tumor lysis assay, the CD276.6second generation, version 1 CAR T cells had high levels of lysisagainst CD276-expressing targets, but also a higher level of activityagainst the negative control cell line.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-29. (canceled)
 30. A polypeptide comprising a heavy and light chaincomprising (i) SEQ ID NOs: 1-6; a heavy and light chain comprising (ii)SEQ ID NOs: 11-16; or a heavy and light chain comprising (iii) SEQ IDNOs: 20-25; wherein the heavy and light chains of (i), (ii), or (iii)bind to CD276, and are linked to one or more of the immunoglobulinconstant domains CH2 and/or CH3.
 31. The polypeptide according to claim1, wherein the one or more immunoglobulin constant domains comprise SEQID NO:
 71. 32. The polypeptide according to claim 30, wherein the heavyand light chains of (i), (ii), or (iii) and the one or moreimmunoglobulin constant domains comprise SEQ ID NO: 80, SEQ ID NO: 82,or SEQ ID NO:
 84. 33. A protein comprising: a first polypeptide chaincomprising (i) a heavy chain comprising SEQ ID NOs: 1-3, (ii) a heavychain comprising SEQ ID NOs: 11-13, or (iii) a heavy chain comprisingSEQ ID NOs: 20-22; and a second polypeptide chain comprising (iv) alight chain comprising SEQ ID NOs: 4-6, (v) a light chain comprising SEQID NOs: 14-16, or (vi) a light chain comprising SEQ ID NOs: 23-25,wherein the first polypeptide of (i), (ii), or (iii) and the secondpolypeptide of (iv), (v), or (vi) bind to CD276 and are linked to one ormore of the immunoglobulin constant domains CH2 and/or CH3.
 34. Theprotein according to claim 33, wherein the one or more immunoglobulinconstant domains comprise SEQ ID NO:
 71. 35. The protein according toclaim 33, wherein the first polypeptide chain, second polypeptide chain,and one or more immunoglobulin constant domains comprise SEQ ID NO: 80,SEQ ID NO: 82, or SEQ ID NO:
 84. 36. The polypeptide according to claim30, further comprising a linker amino acid sequence.
 37. The polypeptideaccording to claim 36, wherein the linker comprises SEQ ID NO:
 115. 38.The polypeptide according to claim 30, comprising SEQ ID NO 10, SEQ IDNO: 19, or SEQ ID NO:
 28. 39. A chimeric antigen receptor (CAR)comprising (a) an antigen binding domain formed by a heavy and lightchain comprising (i) SEQ ID NOs: 1-6, a heavy and light chain comprising(ii) SEQ ID NOs: 11-16, or a heavy and light chain comprising (iii) SEQID NOs: 20-25, wherein the heavy and light chains of (i), (ii), or (iii)bind to CD276; (b) an immunoglobulin constant domain; (c) atransmembrane domain, and (d) an intracellular T cell signaling domain.40. The CAR according to claim 39, wherein the immunoglobulin constantdomain comprises one or more of CH2 and/or CH3 and is effective toextend the antigen binding domain away from a membrane of a CARexpressing cell.
 41. The CAR according to claim 39, wherein theimmunoglobulin constant domain comprises SEQ ID NO:
 71. 42. The CARaccording to claim 39, wherein the transmembrane domain comprises CD8and CD28.
 43. The CAR according to claim 39, wherein the transmembranedomain comprises any one or more of a CD8 amino acid sequence comprisingSEQ ID NO: 29, a CD28 amino acid sequence comprising SEQ ID NO: 30, anda CD8 amino acid sequence comprising SEQ ID NO:
 31. 44. The CARaccording to claim 39, wherein the intracellular T cell signaling domaincomprises one or more of i) CD28, ii) CD137, and iii) CD3 zeta.
 45. TheCAR according to claim 39, wherein the intracellular T cell signalingdomain comprises a CD28 amino acid sequence comprising SEQ ID NO: 32and/or SEQ ID NO:
 35. 46. The CAR according to claim 39, wherein theintracellular T cell signaling domain comprises a CD137 amino acidsequence comprising SEQ ID NO: 33 and/or SEQ ID NO:
 37. 47. A nucleicacid comprising a nucleotide sequence encoding the polypeptide of claim30.
 48. A recombinant expression vector comprising the nucleic acid ofclaim
 47. 49. An isolated host cell comprising the recombinantexpression vector of claim
 48. 50. A population of cells comprising atleast one isolated host cell of claim
 49. 51. The polypeptide of claim30 expressed in one or more host cells for use in treating cancer in amammal.